Craniofacial morphogenesis is a complicated developmental process that is controlled by many genes. In vertebrates, the majority of craniofacial structures are derived from the cranial neural crest (CNC), and abnormalities in CNC development often lead to craniofacial disorders. Such disorders are common in humans, with an occurrence rate of one per 700 newborns and hundreds of different syndromes reported to date. The long-term objective of this proposed research is to understand the molecular bases of CNC development and craniofacial disorders. Toward this objective a transgenic Xenopus tropicalis line that expresses fluorescent reporter in the neural crest lineage (the snail2-egfp line) has been generated for live imaging of CNC development. The goal of the current application is to test the application of this transgenic line in detecting CNC defects and purifyig CNC cells for molecular studies. The application has two specific aims. In the first aim, the snail2-egfp line will be further characterized and assessed for its usage in real-time detection of defects in early and late CNC development. The integration site of the transgene insertion will be mapped, and experiments will be carried out to determine if the transgene has any effect on CNC development or expression of nearby genes. To test if the transgenic line is suitable for real-time detection of aberrant CNC development, deficiencies in the CNC will be induced at different stages and monitored by using high- resolution fluorescence imaging. In the second aim, the snail2-egfp line will be used to isolate high-purity CNC cells for transcriptome analyses. Several methods, including fluorescence activated cell sorting, will be evaluated for their effectiveness in purifying EGFP-labeled CNC cells. Purified CNC cells collected at different developmental stages will be used for cDNA microarray studies to identify genes that are dynamically regulated in premigratory CNC cells. Genes of interest will be selected and their expression patterns will be validated by other molecular techniques; some of these genes will be further tested for their potential functions in CNC development in future studies. Thus the expected outcomes of the proposed research project include identification of new genes that are potentially important for CNC development, as well as establishment of a new methodology for direct detection of CNC defects in live embryos. The successful achievement of these outcomes will provide us new tools and knowledge in craniofacial research.